Use of PCR–RFLP for genotyping 16S rRNA and characterizing bacteria cultured from halibut fry

Small subunit ribosomal genes were explored using PCR–RFLP to facilitate the characterization of bacteria cultured from reared fry of the Atlantic halibut (Hippoglossus hippoglossus). Concern has been expressed about pathogen invasion in larvae lacking a counteracting normal flora that may aid the immune system in producing robust noninfected individuals. In this study, pure cultured representatives of normal flora that were previously found to be antagonistic towards a pathogenic Vibrio sp. were subjected to a whole cell PCR protocol amplifying ~1500 bp of 16S rDNA. Amplified DNA was digested by AluI, BstUI, CfoI, and RsaI, to generate restriction profiles. Before the isolates were characterized, a survey was performed to test the discriminative efficiency of the RFLP. Efficient detection of polymorphism and the resolution of species and subspecies were achieved. Using the RFLP on 103 isolates generated as many as 22 genotypes. Based on the restriction profiles, a taxonomic tree incorporating 19 reference strains was constructed. Partial sequencing found this tree to be dominated by γ-Proteobacteria in clusters of Vibrio-, Pseudomonas-, and Alteromonas-affiliated species. Only nine isolates fell outside these genera, including the three isolates Shewanella alga, Deleya marina, and Marinomonas protea. These species have not previously been reported as halibut flora. The most frequently isolated genotype resembled Vibrio salmonicida.Key words: halibut, Vibrio, RFLP, 16S rDNA, phylogeny.

Asymmetrie Reduction of Synthetic Ketones by Marine Microorganisms

Three strains of bacteria reducing (trifluoroacetyl)ferrocene (3) to optically pure (R)-2,2,2-trifluoro-l-hydroxyethylferrocene (4) and one bacterial strain reducing 3 to (S)-4 of moderate optical purity were isolated from sea-water collected at the coastal areas in Ibaraki prefecture of Japan. The former three strains were identified as Micrococcus lylae, Micrococcus luteus, and Deleya marina and the latter as Bacillus licheniformis. These strains also asymmetrically reduced some other synthetic ketones, e.g., 2,2,2-trifluoroacetophenone (7) and phenyl trimethylsilyl ketone (9). Further screening of microorganisms capable of reducing 3 was done with bacteria isolated from sea-water of the deep sea (Okinawa trough, Japan trench, and Mariana trough) and of the pelagic areas (Indian Ocean and South China Sea). Most of these marine strains preferentially reduced 3 to (R)-4 similar to the coastal strains, but the frequency of finding very highly enantioselective strains (i.e., those forming 4 of > 90% e.e.) was remark­ ably high in several sites of the deep sea and pelagic areas as compared with the coast and terrestrial environment.

Variable expression of gliding and swimming motility in Deleya marina

Surface-associated motility has been observed in the Deleya marina type strain ATCC 25374 (strain 219). Slime tracks and a complex growth pattern, characteristic of gliding motility, developed on semisolid marine-agar motility plates. Cell movement observed by light microscopy consisted of rapid glides and flips by single cells and groups of cells. Following the development of the gliding cell growth pattern, a subpopulation of swimming cells appeared. The variation in motility was random and reversible in subculture. Electron microscopic comparisons of cells of the two motility types showed that gliding cells had no obvious motility organelles, whereas swimming cells had polar flagella. Variable expression of gliding and swimming motility was also observed in D. marina strain 140 (ATCC 27129) and in two other species of the Deleya genus. Key words: gliding, morphological variation, Deleya, biofouling.